JP2014533676A - Composition comprising chicory extract - Google Patents

Abstract

The present invention relates to various factors of weight loss or weight control or weight gain limitation, fat accumulation, fatty liver, hepatic triglyceride level, hypertriglyceridemia, blood glucose level, insulin blood level, insulin resistance and / or metabolic syndrome. It relates to a chicory extract and a composition comprising such an extract, which makes it possible to reduce or limit.

Description

  The invention relates to weight loss or body weight regulation or weight gain limitation, fat accumulation, fatty liver, hepatic triglyceride levels, blood glucose levels, insulin blood levels, insulin resistance, hypertriglyceridemia, prediabetes and / or metabolic syndrome. It relates to a chicory extract and a composition comprising such an extract, which makes it possible to limit the reduction of various factors or their increase.

  More particularly, the extract may exhibit activity against fatty cirrhosis, cardiovascular disease, diabetic complications and / or risk factors that may affect diabetes. Among these risk factors, mention is made of an increase in various factors of overweight, in particular obesity, fat accumulation, fatty liver, hepatic triglyceride level, hypertriglyceridemia, blood glucose level, insulin blood level, insulin resistance and / or metabolic syndrome. obtain.

  The invention also relates to foods or dietary supplements containing such extracts.

  The present invention also relates to a composition for use in the prevention or treatment of metabolic syndrome, obesity, fatty cirrhosis, cardiovascular disease, diabetic complications and / or diabetes.

  In modern society, the risk factors for diseases such as fatty cirrhosis, cardiovascular diseases, diabetic complications and / or diabetes are increasing.

  Known compounds or compositions that can be used to treat these diseases and / or limit one or more of these risk factors may be effective, particularly in weight loss, but are insufficient, Very expensive, exhibits undesirable side effects, has poor sensory properties, can change the color, taste and / or appearance of food and is intended to be incorporated into food It is difficult, and at least some types of foods are insufficiently stable, may exhibit low solubility, may be insufficiently versatile, and can be procured sufficiently stably It may not be and / or derived from precursors that are abundant or have other uses.

  On the other hand, diets for weight control are often successful to some extent. For example, a low-calorie diet can cause temporary weight loss, but has not proven to be effective for people who want to lose weight over a long period of time and want to maintain a constant weight.

  Accordingly, the present invention is directed to solving all or part of the listed problems.

  According to one aspect, the present invention relates to a plant of chicory (Cichorium genus) comprising a phenolic derivative comprising flavonoids which are the main phenolic acid derivatives identified as chicoryic acid, kaphthalic acid and chlorogenic acid and quercetin and luteolin derivatives. Targeted extracts are derived.

The present invention may exhibit all or part of the following advantages:
-Be able to evaluate parts of the plant, in particular leaves, which have been used at a very low cost and have never been used before but can be easily collected,
-Powerful biological effects,
-Food-grade quality extract, and therefore lead to the possibility of use as a preservative (because it is non-toxic or very low) and / or
-Ease of obtaining an activator.

  Other advantages are shown in the following description.

  According to another aspect, the present invention is directed to a composition comprising a chicory plant extract comprising a phenolic acid derivative and a flavonoid derivative and optionally a carrier.

  The composition or extract containing the extract is weight loss or control or restriction of weight gain, fat accumulation, fatty liver, liver triglyceride level, hypertriglyceridemia, hyperglycemia, blood glucose level, insulinemia, insulin resistance It may be intended to allow a reduction or restriction of an increase in various factors of pre-diabetes and / or metabolic syndrome.

  The composition or extract comprising the extract may comprise weight regulation and / or weight gain and / or increased fat accumulation and / or fatty liver and / or high liver triglyceride levels and / or prediabetes and / or hyperglycemia and It can be used in curative or prophylactic treatment of / or hypertriglycerides and / or hyperglycemia and / or hyperglycemia and / or hyperinsulinemia and / or inrin resistance and / or metabolic syndrome.

  The present invention is also directed to a food composition, solid or liquid comprising a chicory plant extract comprising phenolic and flavonoid derivatives and optionally a carrier.

  The present invention also includes chicory plant extracts containing phenolic and flavonoid derivatives and optionally a carrier, in particular, regulating weight loss or weight gain, fat accumulation, fatty liver, liver triglyceride levels, hypertriglyceridemia Foods or dietary supplements or pharmaceuticals or foods to enable the reduction or limitation of the increase in one, some or all of the factors of blood glucose levels, insulinemia levels, insulin resistance and / or metabolic syndrome Intended for the composition.

  According to another aspect of the present invention, the present invention comprises a chicory plant extract comprising phenolic acid derivatives and flavonoid derivatives and optionally a carrier, in particular fatty liver cirrhosis, cardiovascular disease, diabetic complications and / or For use to prevent and / or treat diabetes and / or one or several factors of metabolic syndrome (e.g. high levels of insulin, hypercholesterolemia, hypertension, overweight, especially obesity and hyperglycemia) Of the pharmaceutical composition.

 The invention also includes fatty cirrhosis, cardiovascular disease, diabetic complications and / or diabetes and / or one or several metabolic syndrome factors (e.g., high levels of insulin, hypercholesterolemia, hypertension, overweight, In particular, it relates to a method for preventing and / or treating obesity and hyperglycemia in a subject in need thereof, comprising administering a composition comprising a chicory plant extract of the present invention.

  The composition may be used to adjust the weight of the subject, particularly to help reduce, control or control the weight of the subject.

  The present invention also provides a method for adjusting a subject's weight, in particular for adjusting the subject's weight in need, and in particular for helping to reduce, adjust or adjust the subject's weight, It also relates to a method comprising administering to said subject a composition comprising an extract of a chicory plant.

  Briefly described, the subject may be a human or animal, particularly a mammal.

  In yet another aspect, the present invention is directed to the use of a chicory plant extract comprising a phenolic acid derivative and a flavonoid derivative for the preparation of a medicament or pharmaceutical composition.

  In the following further embodiments, the present invention is notably limited in weight loss or weight gain, fat accumulation, fatty liver, hepatic triglyceride levels, hypertriglyceridemia, blood glucose levels, insulinemia, insulin resistance and / or metabolic syndrome. The present invention is directed to a diet that allows for the reduction or limitation of an increase in various factors, including ingesting a composition comprising a chicory plant extract comprising a phenolic acid derivative and a flavonoid derivative at least once a day.

  The present invention also limits or reduces various factors of fat accumulation, fatty liver, hepatic triglyceride levels, hypertriglyceridemia, blood glucose levels, insulinemia, insulin resistance and / or metabolic syndrome in a subject in need thereof. And a method comprising administering to the subject a composition comprising an extract of a chicory plant of the present invention.

The chicory (Cichorium genus) plant extracts of the present invention are derived from the following species and mixtures thereof.
-Cichorium intybus L,
-Cichorium intybus var.foliosum,
-Cichorium intybus var.sativum,
-Cichorium intybus ssp. Intybus var. Sativum and
-Cichorium endivia L.

  The chicory plant is preferably selected from Cicholium intybus species, in particular sativum and Cicholium endivia species and mixtures thereof.

  The extract is obtained in particular from the aerial part of the plant, more particularly from the leaves. Thus, the extract may advantageously be a leaf extract.

  The expression “mass of chicory plant extract” or “mass of extract” means, in the sense of the present invention, the dry mass of a chicory plant extract or the dry mass of an extract.

  The chicory plant extract according to the present invention comprises a phenolic acid derivative and a flavonoid derivative. The phenolic acid derivatives advantageously comprise three main phenolic acid derivatives identified as chicory acid, kaphthalic acid and chlorogenic acid. The flavonoid derivatives are advantageously quercetin and luteolin derivatives, in particular quercetin 3-O-glucuronide, isoquercetin and luteolin 7-O-glucuronide.

  The amount of chicory acid in the extract is advantageously greater than or equal to 25 g (25 g / kg) per kg of extract, more particularly greater than or equal to 30 g (30 g / kg) per kg of extract, and even more particularly 45 g per kg of extract ( 45g / kg) or more. In certain embodiments, the amount of chicory acid in the extract depends primarily on the chicory used in the preparation of the extract and can be up to about 65-70 g / kg. In general, the amount of chicory acid in the extract ranges from 45 to 55 g / kg.

  The amount of kaphthalic acid in the extract is advantageously at least 2.5 g (2.5 g / kg) per kg of extract, more particularly at least 4 g (4 g / kg) per kg of extract, even more particularly per kg of extract It is 5g (5g / kg) or more. In certain embodiments, the amount of kaphthalic acid in the extract depends primarily on the chicory used in the preparation of the extract and can be up to about 10 g / kg. Generally, the amount of kaphthalic acid in the extract is in the range of 5-6 g / kg.

  The amount of chlorogenic acid in the extract is advantageously greater than or equal to 1 g (1 g / kg) per kg of extract, more particularly greater than or equal to 1.5 g (1.5 g / kg) per kg of extract and even more particularly per kg of extract 2g (2g / kg) or more. In certain embodiments, the amount of chlorogenic acid in the extract depends primarily on the chicory used in the preparation of the extract and can be up to about 10 g / kg. Generally, the amount of chlorogenic acid in the extract is in the range of 5-6 g / kg.

  The extract is the combined amount of kaphthalic acid and chicory acid, more than 30g (30g / kg) per kg of extract, more particularly more than 40g (40g / kg) per kg of extract, and even more particularly 50g per kg of extract (50 g / kg) or more, preferably in the range of 50-60 g / kg.

  The weight ratio of chicory acid / caphthalic acid may be 2-20, in particular 3-15, more particularly 4-14, even more particularly 6-12, preferably 7-11.

  The mass ratio of chicory acid and caphalic acid / chlorogenic acid may be from 5 to 60, in particular from 6 to 40, more particularly from 7 to 50, even more particularly from 8 to 20, preferably from 8 to 19.

  The amount of flavonoid derivative in the extract is expressed in accordance with the standard for quercetin, preferably 4.5 g (4.5 g / kg) or more per kg of extract, more particularly 5 g (5 g / kg) or more per kg of extract And more particularly 6.5 g (6.5 g / kg) or more per kg of extract, preferably in the range of 6.5 to 10 g / kg.

  The amount of flavonoid derivative may be determined based on the UV spectrum using quercetin as a standard. That is, the amount of flavonoids is the mass of the flavonoids, in particular the mass of the quercetin derivative expressed corresponding to the quercetin standard.

  The extract advantageously comprises a total phenol content, which is a total phenol content of 3-10% by weight, corresponding to gallic acid.

  The total phenol content in the extract may be determined using Folin-Ciocalteu reagent and / or further by reverse phase HPLC.

  The method of preparing the extract of the present invention from chicory biomaterial must follow a suitable method to result in an active extract containing phenolic and flavonoid derivatives as defined above and in the examples.

  In particular, the method must take into account the stability of phenolic acid derivatives and flavonoid derivatives. Since phenolic acid derivatives and flavonoid derivatives are sensitive to polyphenol oxidase, purification of the extract must be performed taking into account the sensitivity of the phenol derivative to certain reactants such as polyphenol oxidase. Those skilled in the art will identify these components to be avoided in the extraction and purification steps based on their technology and knowledge and the disclosure of specific embodiments of the invention.

  The extract according to the invention is preferably an aqueous or hydroalcoholic extract, in particular a water ethanol extract. The extract may in particular be an extract obtained or obtainable by the method disclosed in the description herein.

  The invention also relates to a composition comprising a chicory plant extract as described above and below and optionally a carrier.

  The carrier may in particular be an edible carrier, usually used for pharmaceutical compositions, nutritional supplements and foods or dietary supplements. The carrier may include one or more flavoring agents.

  The composition comprises 10 to 99%, more preferably 25 to 95%, even more preferably 50 to 95%, and even more preferably 75% chicory plant extract, based on the total weight of the composition. It may be included in an amount of ~ 95% by weight.

  The food or dietary supplement may comprise the extract in an amount ranging from 1 to 100% by weight, based on the total weight of the dietary supplement.

  The nutritional supplement composition or food composition may comprise the extract in an amount ranging from 0.1 to 5% by weight, based on the total weight of the composition.

  The dietary supplement, dietary supplement composition or food composition may be liquid, solid or powder.

  The composition may be formulated to allow a human to take in the range of 5-60 mg / kg, especially about 30 mg / kg per day.

The invention also relates to a method as defined above and hereinafter for preparing an extract of a chicory plant, the extraction method comprising the following steps:
a) contacting the ground chicory plant with an extraction solvent;
b) removing the solid content by filtration and collecting the solvent extract,
c) a step of optionally washing the solid content by stirring with an extraction solvent,
d) optionally removing the solids by filtration and collecting the solvent extract;
e) combining the solvent extracts to remove insoluble residues;
f) concentrating the solvent;
g) a step of concentrating the phenolic compound optionally contained in the extract by precipitation, in particular using food grade alcohol;
h) filtering and evaporating the filtrate; and
i) recovering the dried extract.

  The extraction method of the present invention is preferably carried out on the aerial parts of plants, more preferably on leaves.

  The plant may be used in an undried state, after blanching, or dried. Preferably, undried leaves of dried or blanched chicory plants are used in step a).

  The extraction of step a) is advantageously carried out at a temperature above 25 ° C., in particular above 35 ° C. or more particularly above 50 ° C.

  In particular, the extraction of step a) is advantageously carried out at a temperature above 25 ° C., more particularly at a temperature above 35 ° C. or even more particularly at about 50 ° C.

  Advantageously, the extraction of step a) is carried out at a temperature of 25 to 70 ° C., preferably 40 to 60 ° C.

  The solvent for the extraction step is preferably selected from aqueous, alcohol, water-soluble organic solvents and combinations thereof. The water soluble organic solvent may be acetone.

  The resulting extract may be considered an aqueous, alcohol or organic solvent extract.

  Suitable solvents may be selected from water, methanol, ethanol, acetone, n-propanol, isopropanol, 2-butanol and combinations thereof.

  More particularly, the extraction solvent comprises at least 75% by weight, at least 90% by weight, more particularly at least 95% by weight, and even more particularly at least 99% by weight, based on the total weight of the extraction solvent, more preferably, Consists of these solvents.

  In the following embodiments, the extraction is carried out with water and / or an alcohol solvent, especially ethanol.

  When extraction is carried out with water and alcohol, in particular ethanol, the volume ratio of water / alcohol is 80/20 to 20/80, in particular 70/30 to 30/70, more particularly 60/40 to 40/60 and even more particularly It may be about 50/50.

  The proportion of alcohol, especially ethanol, used in the extraction can affect the yield and composition of the biologically active compound.

  Extraction can be performed in a batch or continuous manner, or by continuously using the same extraction solvent in a continuous batch to obtain a saturated solvent for the extract. For batch extraction, one skilled in the art will define an appropriate mass ratio of solvent / plant solids optimized in an industrial process. The solvent / solids ratio may be 2 to 20 times, in particular 5 to 15 times, more particularly about 10 times, relative to the solids mass. The extraction may be performed once or several times, in particular 2 to 4 times.

  In a continuous or continuous batch extraction process, it may be used at a low rate.

  The process according to the invention may be carried out in the range of 25-70 ° C., in particular 40-60 ° C. and more particularly at about 50 ° C.

  The extraction may be continued with stirring (eg mechanical or magnetic stirring) for 1 to 5 hours, especially about 2 hours.

  The remaining solids may be removed, particularly by filter filtration with a filter bag.

  According to the invention, several extractions may be a combination of single extractions.

  The wet solids may be further extracted with a solvent, in particular a hydroalcoholic mixture, at 1 to 100 times the mass of dry solids and further stirred for about 10 to 120 minutes.

  Solids may be collected and the extracts combined.

  Different extract solutions may be combined, left for decantation, and filtered by filter paper or centrifugation to remove insoluble residues.

  The resulting clear supernatant may be concentrated to about 5-20% of the initial volume, for example using a concentrator, and then with a food grade alcohol, especially ethanol, at a certain rate (e.g., at a minimum). Treatment with twice the concentrated volume) to remove the precipitate formed. This step may allow removal of all or part of the undesired compound (eg polysaccharide or water-soluble protein).

  The powder extract may be obtained by drying the concentrated extract using, for example, a spray dryer, an oven at 50-80 ° C. or a vacuum dryer.

The dry extract is weighed (g) and the yield of the extract is calculated by the following formula:
Dry yield (%) = (dry weight of extract / dry weight of plant) x 100
Undried yield (%) = (dry weight of extract / undried weight of plant material organism) x 100

More particularly, the plant extract may be obtained by a method comprising the following steps:
a) The ground and compressed plant is extracted with 2 to 20 times, in particular about 10 times the amount of extraction solvent (especially at least 30% by volume ethanol-water mixture, more particularly ethanol-water 1/1). An ethanol-water mixture containing ethanol in a volume ratio of) and stirring for example for 2 hours, in particular at a temperature higher than 25 ° C., more particularly at a temperature higher than 35 ° C. and even more particularly at about 50 ° C.,
b) a step of removing the solid content by filtration to recover the solvent extract;
c) washing the wet solids by stirring with the extraction solvent, in particular in an amount corresponding to 1 to 100 times the mass of the dry solids, for example for about 15 to 120 minutes, in particular 15 to 30 minutes,
d) collecting the solvent extract by filtering the solids;
e) combining the extraction solutions and removing insoluble residues by decanter or filter filtration;
f) concentrating the extract by concentrating the solvent and precipitating in ethanol;
g) concentrating the filtrate under reduced pressure; and
h) drying (e.g. placing in an oven at 50-80 <0> C under vacuum, e.g. vacuum spray drying or the concentrate may be frozen for lyophilization), and
i) recovering the dried extract.

  In general, the yield of the extract is in the range of 1.5 to 3.5% by weight of the extract, based on the total weight of the crushed solids that have not been dried. When using dry leaves, the yield ranges from 12 to 17%.

  Plant extracts can be prepared on a commercial scale by repeating the extraction process to isolate the target extract.

  Thus, a small scale extraction method can be scaled up with additional quality control steps optionally included to ensure reproducible results of the resulting extract.

  Various extraction methods can be employed. In general, the extract is sufficient to bring the solid plant into contact with the solvent so that the biologically active molecules present in the plant material are incorporated into the solvent. Obtained by contacting for a period of time to ensure exposure.

  The solvent extraction method may be selected from direct and continuous (countercurrent) extraction with polar and / or non-polar solvents at room temperature or elevated temperature. Sufficient contact of the solvent with the plant material can be facilitated by shaking the suspension. The liquid fraction is then separated from the solid (insoluble) fraction, resulting in two fractions: a liquid fraction that becomes an extract and a solid fraction.

  Separation of liquid and solid fractions can be achieved by one or more standard methods known to those skilled in the art.

  The present invention also relates to the sensitivity of the extract obtained or obtainable by the method of the present invention, said extract comprising a phenolic acid derivative and a flavonoid derivative.

FIG. 1 shows an HPLC chromatogram at 280 nm of a hydroalcoholic plant extract. FIG. 2 shows the effect of the chicory extract of the present invention on blood glucose in obese prediabetic mice. FIG. 3 shows the effect of the chicory extract of the present invention on blood insulin and insulin resistance in obese prediabetic mice. FIG. 4 shows the effect of the chicory extract of the present invention on body weight and fat accumulation in obese prediabetic mice. FIG. 5 shows the effect of the chicory extract of the present invention on fatty liver or hepatic steatosis in prediabetic mice. FIG. 6 shows the dose-dependent effect of the chicory extract of the present invention on blood glucose in obese prediabetic mice. FIG. 7 shows the effect of the chicory extract having a low concentration of chicory acid and the chicory extract of the present invention on the blood glucose level in obese diabetic mice. FIG. 8 shows the effect of purified chicory acid extract on blood glucose levels in obese prediabetic mice. FIG. 9 shows the effect of Echinacea leaf extract and chicory extract of the present invention on blood glucose level in obese prediabetic mice. FIG. 10 shows effects of Echinacea leaf extract and chicory extract of the present invention on blood insulin and insulin resistance in obese diabetic mice.

  (Example 1: Preparation of chicory (Cichorium intybus L) extract)

Materials and Methods Dried aboveground parts (100 g) of chicory (Cichorium intybus L) are shredded (5-8 mm) before extraction.

Extraction method Plants are extracted with about 1000 ml of 50% ethanol / water (V / V) under mechanical stirring for 2 hours at 50 ° C. At the end of the extraction time, the solids are removed by filtration through a filter bag (PE-100) or other similar filter media. The wet solids are extracted once more by stirring with another 3 volumes of 50% ethanol for an additional about 15-30 minutes.

  Solids are again filtered off and the resulting extracts are combined to give 980 ml of liquid extract, left for decantation and filtered or filtered through filter paper to remove insoluble residues. except.

  The extraction solution was concentrated under reduced pressure to 5-20% of the initial volume and concentrated by precipitation with food grade ethanol. The filtrate is concentrated again and subjected to drying. Collect the dry extract.

  The HPLC chromatogram of the plant extract of Example 1 at 280 nm is shown in FIG. 1 as chromatogram I (A is caphthalic acid, B is chlorogenic acid, C is chicory acid, D is quercetin 3-O-glucuronide and isoquercetin, and E Is luteolin 7-O-glucuronide). Further, the composition of the extract is shown in Table 1 below.

  (Example 2: Preparation of chicory (Cichorium intybus ssp intybus var. Sativum) extract)

  The same method as in Example 1 was followed except that the above-ground part of undried chicory (Cichorium intybus ssp intybus var. Sativum) was used and boiled before the shredding step.

  An HPLC chromatogram at 280 nm of the plant extract of Example 2 is shown in FIG. 1 as Chromatogram II ((A is caphthalic acid, B is chlorogenic acid, C is chicory acid, D is quercetin 3-O-glucuronide and isoquercetin and E is luteolin 7-O-glucuronide) and the composition of the extract is shown in Table 1 below.

  (Example 3: Composition of the extract obtained from Examples 1 and 2)

Analytical methods The extracts from Examples 1 and 2 were equivalent to gallic acid and contained a total phenol content of 3-7% by weight using the foreign thiocult method.

Characterization and quantification of polyphenol content by HPLC
-HPLC-DAD analysis:
Requires analytical HPLC (Dionex) setup. In the present invention, the mobile phase used was 0.1% formic acid (solvent A) and acetonitrile (solvent B) in 1000 ml of high-purity water, and the total run was performed at a flow rate of 0.8 ml / min for 96 minutes using the following gradient. did. The slope points are: time 0.0 minutes (A 95% and B 5%); 10 minutes (A 90% and B 10%); 10 minutes (constant composition) and 20-40 minutes (B 10-20) The gradient changes linearly to%); again for 10 minutes (constant composition); 50 to 65 minutes (B varies linearly to 20-30%) and the next 10 minutes (linear to 50%) 75-76 minutes (the slope changes linearly from 50 to 100%) and 10 minutes (constant composition), 85-86 minutes are the first conditions (95% A and 5% B) %) At a constant composition for 10 minutes. Phenolic compounds in the eluate were detected with a UV-diode array at 280 nm using a reverse phase C-18 column (250 × 4.6 mm ID × 5 μm; ACE). The amount of phenolic acids in the extract was determined using a calibration curve of kaphthalic acid; chlorogenic acid; chicory acid; and the amount of flavonoids in the extract was determined using a quercetin calibration curve.

-HPLC-DAD / ESI-MS analysis:
HPLC / MS analysis of the extract was performed using HPLC (Thermo Finnigan surveyor) and interfaced to an LCQ ion trap spectrometer fitted with an electrospray interface (Thermo Finnigan, LCQ Advantage max). The elution program was the same as described above, and the experiment was performed in both positive and negative modes. The spectrum was scanned over the mass range m / z 80-2000.

Quantified composition of the extracts of Examples 1 and 2 by HPLC at 280 nm

  (Example 4: Effect of chicory leaf extract (CLE) on blood sugar (blood sugar), blood insulin (insulin blood), insulin resistance, body weight, fat accumulation and fatty liver)

Materials and methods
Five-week-old C57BL / 6N1 Crl mice (Charles River Laboratories, France), weighing approximately 20 g, were used for the experiment. After 8 days of acclimatization, mice were randomly assigned to different experimental groups (9 per group) according to fasting blood glucose.

On day 0, place one group under normal diet (23% of calories are protein-derived, 66% are carbohydrate-derived and 11% fat-derived), and the other group is a high fat diet (17% of calories are protein And 28% derived from carbohydrate and 55% derived from fat). Food and water were given freely. A single prescription was administered by oral gavage every morning between 9 and 10 o'clock from day 1 to day 56. The administration volume was 10 ml / kg body weight (10 ml / kg). The actual volume to be administered was calculated and adjusted based on the latest body weight of each animal individual. The test conditions are as follows:
Normal control: Mice were fed a normal diet and given water once a day.
High fat control: Mice were fed a high fat diet and given water once a day.
CLE 400 mg / kg: A high-fat diet was given to mice, and chicory leaf extract (CLE) was administered once a day at a dose of 400 mg / kg body weight (400 mg / kg).

  FIG. 2 shows the effect of CLE on blood glucose, FIG. 3 shows the effect on insulin and insulin resistance, FIG. 4 shows the effect on body weight and fat accumulation, and FIG. 5 shows the effect on fatty liver.

  Mouse weights were recorded upon arrival and three times a week thereafter (Monday, Wednesday and Friday)

  Fasting blood glucose levels were fasted for 4 hours between 13:00 and 14:00 on the day of randomization (day 0) and on days 7, 14, 21, 28, 35, 42, 49 and 56 Measured on animals. A whole blood sample (1 drop) was collected from the tail vein and blood glucose was determined with a hand-held blood glucose meter (OneTouch Ultra 2, LifeScan).

  At the end of the study, the animals were anesthetized with 0.1 ml of pentobarbital sodium administered intraperitoneally after being fasted for 4 hours. The final blood sample was collected by cardiac puncture using heparin as an anticoagulant. This final blood sampling causes the animal to die. Blood samples were collected and placed at 4 ° C. and centrifuged for 30 minutes, and plasma was collected and frozen until insulin analysis. Epididymal fat pad (abdominal fat) and liver were collected and weighed.

Fasting plasma insulin levels were measured with the ELIZA kit (Mercodia, Sweden). The insulin resistance index (HOMA-IR) is then expressed as: HOMA-IR = fasting insulin (mU / l) x fasting glucose (mmol / l) /22.5
Calculated using

  This protocol was approved by the Ethics Committee (Montpellier, France).

Results blood glucose (Figure 2)
After one week under diet (Day 7), fasting blood glucose (168 ± 5.7 mg / dL) in mice fed a high fat diet is the blood glucose level (121 ± 5.9 mg / dL) in mice fed a normal diet It was significantly higher than. Since then, this difference has remained constant throughout the survey.

  Chicory leaf extract reduced blood glucose in prediabetic mice. At the end of the study (day 56), blood glucose dropped by 58%. This effect was clearly significant on days 14, 28, 35, 42, 49 and 56.

Blood insulin and insulin resistance (Figure 3)
Fasting insulinemia also increased significantly to 1.70 ± 0.43 ng / ml with a high fat diet, compared to 1.53 ± 0.37 ng / ml in mice under normal diet. As the fasting blood glucose and insulin blood increased with a high fat diet, the insulin resistance index (HOMA-IR) increased 5-fold, indicating that these animals are strongly insulin resistant.

  At the end of the treatment period (day 56), CLE significantly reduced fasting insulin levels induced by a high fat diet (p <0.05). Insulinemia reached 3.31 ± 0.40 ng / ml in CLE-treated mice. Thus, according to the HOMA-IR insulin resistance index, CLE reduced insulin resistance induced by high fat diet in prediabetic mice by 58% (p <0.01).

Body weight and abdominal fat mass (Figure 4)
Mice fed a high fat diet gained much more weight than mice fed a normal diet. At the end of the study, after eating for 8 weeks, mice on a high fat diet gained 2.5 times the body weight compared to control mice on a normal diet. The normal diet mice reached 26.3 ± 0.5 g, while the high fat diet mice reached 36.8 ± 1.3 g.

  The weight gain due to meals is due in particular to storing energy as abdominal fat. Certainly, the mass of epididymal (abdominal) fat increased 3.5 times during the study period in mice under high fat diet compared to mice under normal diet (high fat diet mice and normal diet mice 1.80 ± 0.11 and 0.51 ± 0.03 g respectively). Peri-visceral fat is recognized to be or is a strong risk factor for insulin resistance and metabolic syndrome.

  CLE at a dose of 400 mg / kg body weight (400 mg / kg) reduced the increased body weight on a high fat diet. This effect was significant from day 4 of treatment and persisted throughout the study period (p <0.01 after day 6). At the end of the study, there was an increase of 10.1 ± 0.8 g in mice treated with CLE on a high fat diet, while an increase of 15.4 ± 1.3 g in mice treated with water on a high fat diet, as well as water on a normal diet. In the treated mice, the increase was 6.1 ± 0.4 g.

  This decrease is due, at least in part, to a decrease in fat accumulation in abdominal fat. CLE reduced fat accumulation of epididymal fat in mice subjected to a high fat diet by 33% (p <0.05). The mass of epididymal fat in CLE-treated mice was 1.38 ± 0.15 g versus 1.80 ± 0.11 and 0.51 ± 0.03 g in the high fat diet control and normal diet control, respectively.

Liver mass (Figure 5)
A high fat diet induced fat accumulation in the liver (so-called fatty liver or liver steatosis). The color of the liver became white instead of red in mice on a normal diet, and its weight increased by 35% (1.38 ± 0.10 g on a high fat diet, 1.02 ± 0.03 g on a normal diet).

  CLE decreased fatty liver by 83% and was almost normalized (p <0.01).

Conclusions High-fat diet induces a clear phenotype of metabolic syndrome in C57 / BL6 mice: significant weight gain, abdominal fat accumulation, fatty liver, fasting glycemia and insulinemia, and insulin resistance .

A chicory leaf extract at a dose of 400 mg / kg body weight (400 mg / kg) significantly reduced abnormalities induced by chronic high-fat diet intake in mice. It includes:
-Decrease in fasting blood sugar
-Decreased fasting of insulin blood
-Reduced insulin resistance
-Weight loss
-Reduced abdominal fat accumulation
-Reduced liver steatosis

  Thus, chicory leaf extract has been shown to provide good health, good composition, and address one or several metabolic syndromes and related abnormalities.

(Example 5: Dose-dependent effect of chicory leaf extract (CLE) on blood glucose (glycemia))
The material and method protocol was the same as in Example 4.
The experimental conditions were as follows:
-Normal control: Mice were fed a normal diet and given water once a day.
-High fat control: mice were fed a high fat diet and given water once a day.
-Chicory leaf extract (CLE) 100, 200 or 400 mg / kg: Give the mouse a high-fat diet and once daily chicory leaf extract (CLE) at a dose of 100, 200 or 400 mg / kg body weight Administered.

Results blood glucose (Figure 6)
After one week under diet (day 7), fasting blood glucose (159 ± 3.9 mg / dL) in mice fed a high fat diet is the blood glucose level (109 ± 3.6 mg / dL) in mice fed a normal diet It was significantly higher than. Since then, this difference has remained constant throughout the survey.

  Chicory leaf extract reduced blood glucose in prediabetic mice on days 14-42. The dose of CLE increases this effect and is significant during the three doses of the study. At the end of the study, blood pressure reached 187 ± 3.2, 177 ± 3.1, and 171 ± 6.3 mg / dL for doses 100, 200, and 400 mg / kg, respectively, compared to 200 ± 5.4 mg / dL in the control. .

Conclusions Chicory leaf extract (CLE) apparently dose-dependently reduced fasting hyperglycemia induced by chronic high fat intake in mice. This effect is already significant in the low dose study (100 mg / kg body weight).

(Example 6: Chicory leaf extract (CLE) containing low levels of chicory acid has a lower effect on lowering blood glucose)
Materials and Methods Low chicory acid-CLE extraction method Low chicory acid-CLE was extracted according to the extraction method described in Example 2 except that the blanching step was not performed prior to the shredding step. As a result, plant polyphenol oxidase was inactive and polyphenol levels were low. Low chicory acid-CLE contained 1.05% chicory acid.

CLE-extraction method
CLE was extracted according to the method described in Example 2. CLE contained 4.68% chicory acid.

The pharmacological protocol protocol is the same as in Example 4, but C57BL / 6 mice were subjected to a high fat diet for one week before the start of treatment, leading to pre-diabetes. CLE and low chicory acid-CLE were then given by oral gavage at a dose of 200 mg / kg body weight (200 mg / kg) for 3 weeks.

Results Blood sugar in fasting conditions during fasting (Figure 7)
After a week on the diet, fasting blood glucose (159.6 ± 4.9 mg / dL) in mice fed a high fat diet was compared to that in mice fed a normal diet (95.2 ± 4 mg / dL). Significantly higher (p <0.001). This result confirms that mice on a high fat diet were hyperglycemic at the start of treatment.

  The effect of CLE extract on reducing blood glucose in prediabetic mice by an average of 31.5% (relative to the normal value observed in normal controls) was clearly significant from the first week of treatment and was the longest investigated (Figure 7: * p <0.05 on day 7 and *** p <0.001 on days 14 and 21).

  On the other hand, low chicory acid-CLE showed a much lower effect. In prediabetic mice, blood glucose decreased by an average of 10.6%. This effect was significant on day 14 but not on days 7 and 21 (FIG. 7: * p <0.05).

Conclusion
C57BL / 6 mice were subjected to a high fat diet for one week before hyperglycemia. The dose of 200 mg / kg body weight (200 mg / kg) of chicory leaf extract (CLE; containing 4.68% chicory acid) clearly reduced hyperglycemia. This effect is associated with the presence of a sufficient amount of chicory acid in the extract, as low chicory acid-CLE (containing 1.05% chicory acid) is less effective.

  Therefore, chicory acid is one of the components of CLE important for the effect of the extract. A sufficient amount of chicory acid extract is necessary to observe the hypoglycemic effect.

(Example 7: Purified chicory acid (25%) has no effect on lowering blood glucose)
Materials and Methods Purified chicory acid (25%)
Purified chicory acid was obtained from a commercial source. In short, polyphenols extracted from plant sources were purified with an adsorbent resin to obtain a product rich in polyphenols and containing about 25% chicory acid.

The pharmacological protocol protocol was the same as in Example 4, but C57BL / 6 mice were subjected to a high fat diet for one week before the start of treatment to induce hyperglycemia. Thereafter, 25% purified chicory acid was administered by oral gavage at a dose of 200 mg / kg body weight (200 mg / kg) for 8 weeks.

Results Effects on fasting blood glucose (Figure 8)
In contrast to CLE, purified chicory acid (25%) had no effect on fasting blood glucose in prediabetic mice after 8 weeks of treatment (FIG. 8).

Conclusion Therefore, even if chicory acid is an essential component for CLE efficacy, it is not sufficient. Cofactors that are suppressed in chicory acid purification are also important for the effect of CLE in reducing fasting blood glucose in prediabetic mice.

(Example 8: Chicory leaf extract (CLE) is more potent than Echinacea leaf extract (EchLE))
Materials and Methods Echinacea Leaf Extract (EchLE)-Extraction Method
EchLE was extracted according to the extraction method described in Example 1. EchLE contained 3.32% chicory acid.
Chicory leaf extract (CLE)-extraction method
CLE was extracted according to the extraction method described in Example 2. CLE contained 4.68% chicory acid.

The pharmacological protocol protocol is the same as in Example 6. Prior to the start of treatment, C57BL / 6 mice were subjected to a high fat diet for one week to induce prediabetes. Thereafter, CLE and EchLE were administered by oral gavage at a dose of 200 mg / kg body weight (200 mg / kg) over 3 weeks.

Results Effects on fasting blood glucose (Figure 9)
CLE extract reduced blood glucose in prediabetic mice by an average of 31.5% (relative to normal values observed in normal controls). This effect was clearly significant from the first week of treatment and lasted up to the end of the study (Figure 9: * p <0.05 on day 7 and *** p <0.001 on days 14 and 21) .

  EchLE showed a much lower effect. EchLE reduced blood glucose in prediabetic mice by an average of 13.1%. This effect was significant on day 21 (FIG. 9: p <0.05), but not significant on days 7 and 14.

Blood insulin resistance (Figure 10)
At the end of the treatment period (21 days), fasting insulin blood was also significantly increased by the high-fat diet compared to 2.43 ± 0.20 ng / ml in mice on a normal diet, 5.26 ± 0.27 ng / ml Reached. With this increase in fasting blood glucose and insulin blood, the insulin resistance index (HOMA-IR) increased three-fold, and at the end of the protocol the mice showed clear insulin resistance.

  CLE significantly reduced fasting blood insulin levels induced by a high fat diet to 4.16 ± 0.23 ng / ml (FIG. 10: ** p <0.01). Thus, according to the insulin resistance index (HOMA-IR), CLE reduced insulin resistance induced by a high fat diet in prediabetic mice by 45.9% (FIG. 10: *** p <0.001) .

  Under the same conditions, EchLE slightly reduced insulinemia (4.16 ± 0.24 ng / ml), but this effect is not statistically significant. Insulin resistance decreased by 18.7% but was not significant (FIG. 10).

Conclusion Chicory leaf extract (CLE; containing 4.68% chicory acid) is prominent in hyperglycemia, hyperinsulinemia and insulin resistance in prediabetic mice at a dose of 200 mg / kg body weight (200 mg / kg) Lowered to. This effect is related to the presence of a sufficient amount of chicory acid in the extract, but Echinacea leaf extract (EchLE), which contains a very high amount of chicory acid (3.32% chicory acid), is less effective. It is also associated with chicory sources. Cofactors present in chicory plants but not in Echinacea are important for CLE effects.

  As mentioned above, this result shows that the chicory acid and cofactor of the extract derived from chicory are important for the effect of CLE.

Claims (20)

  An extract of a chicory plant comprising chicory acid, caphthalic acid and chlorogenic acid, quercetin and / or luteolin derivatives and optionally a carrier, wherein the amount of chicory acid is 25 g / kg or more based on the extract.   The extract according to claim 1, wherein the amount of the kaphthalic acid is 2.5 g / kg or more based on the extract.   The extract according to claim 1 or 2, wherein the amount of chlorogenic acid is 1 g / kg or more based on the extract.   The extract according to any one of claims 1 to 3, wherein the amount of the flavonoid derivative in the extract is expressed in correspondence with a quercetin standard and is 4.5 g / kg or more based on the extract. a) contacting the ground chicory plant with an extraction solvent;
b) removing the solids by filtration and collecting the solvent extract;
c) a step of optionally washing the solid content by stirring with an extraction solvent,
d) optionally removing the solids by filtration and collecting the solvent extract;
e) combining the solvent extracts to remove insoluble residues;
f) concentrating the solvent;
g) a step of concentrating the phenolic compound optionally contained in the extract by precipitation, in particular using food grade alcohol;
h) filtering and evaporating the filtrate; and
i) recovering the dried extract;
A process for preparing an extract as defined in any one of claims 1 to 4 carried out in a temperature range of 40-60 ° C.   6. The method of claim 5, wherein the chicory plant is selected from the group consisting of Cicholium intybus species plants, Cicholium endivia species plants and mixtures thereof.   The method according to claim 5 or 6, wherein the above-ground part of the plant is used.   The method according to any one of claims 5 to 7, wherein the plant is dried, undried or boiled.   9. The method according to any one of claims 5 to 8, wherein the extraction solvent comprises water and alcohol in a water / alcohol volume ratio in the range of 80/20 to 20/80.   An extract of a chicory plant comprising a phenolic acid derivative and a flavonoid derivative as defined in any one of claims 1 to 4, or an extract obtained by the method as defined in any one of claims 5 to 9, And optionally a carrier.  Extract according to any one of claims 1 to 4, for use in curative or prophylactic treatment of body weight and / or regulation of weight gain, as defined in any one of claims 5 to 9 11. An extract obtained by the method or the composition according to claim 10.   Extract according to any one of claims 1 to 4, for use in curative or prophylactic treatment of fatty accumulation and / or fatty liver, as defined in any one of claims 5 to 9. 11. An extract obtained by the method according to claim 10, or a composition according to claim 10.   The extract according to any one of claims 1 to 4 for use in curative or prophylactic treatment of high liver triglyceride levels, obtained by the method as defined in any one of claims 5 to 9. Or a composition according to claim 10.   The extract according to any one of claims 1 to 4 for use in the curative or preventive treatment of hypertriglyceridosis, obtained by the method as defined in any one of claims 5 to 9. 11. An extract or composition according to claim 10.   The extract according to any one of claims 1 to 4 or the method according to any one of claims 5 to 9 for use in curative or preventive treatment of hyperglycemia. 11. An extract or composition according to claim 10.   Obtained by an extract according to any one of claims 1 to 4 or a method as defined in any one of claims 5 to 9 for use in curative or preventive treatment of hyperinsulinemia. Or a composition according to claim 10.   10. An extract according to any one of claims 1 to 4 or a method as defined in any one of claims 5 to 9 for use in curative or prophylactic treatment of insulin resistance. 11. An extract or composition according to claim 10.   The extract according to any one of claims 1 to 4 or the method according to any one of claims 5 to 9 for use in curative or prophylactic treatment of pre-diabetes. 11. An extract or composition according to claim 10.   The extract according to any one of claims 1 to 4 or the extract obtained by the method according to any one of claims 5 to 9 for use in curative or prophylactic treatment of metabolic syndrome. Or a composition according to claim 10.   11. The extract according to any one of claims 1 to 4, or the extract obtained by the method as defined in any one of claims 5 to 9, or the composition according to claim 10 and optionally a carrier. A food or nutritional supplement, a nutritional supplement or food composition.